Germ granules are evolutionary conserved ribonucleoprotein complexes necessary for fertility present in animal germ cells. The full complement of their components is not known in any organism, but many of discovered components are conserved from C. elegans, to Drosophila, to mouse and human. This proposal focuses on identifying new components and regulators of germ granule function in a model system C. elegans. This will be carried out by a combination of genetic screening and proteomic approaches. First, I will use RNAi to functionally screen the germline-expressed genes and assess localization of fluorescently tagged P granules of C. elegans. For this visual screen, I will use two reagents already available in the Seydoux lab: a strain of C. elegans expressing a GFP fusion to the germ granule component PGL-1, and an RNAi library targeting -3,000 genes expressed preferentially in the female germline. In a second approach, I will use biochemical methods to isolate P granule complexes. I will tag P granules in select germ cell sub- types (adult gonadal stem cells and embryonic primordial germ cells) by generating transgenic worms expressing tagged P granule components PGL-1 and GLH-1 in the germline under the control of defined regulatory elements. This will permit biochemical purification of populations of P granules specific to mitotic germline of embryonic primordial germ cells. The protein components of the isolated complexes will be identified by mass-spectrometry. Validity of candidate interactors will be assessed by alternative methods, such as yeast two-hybrid assays or in-vitro GST-pulldown assays. Positives resulting from both RNAi and proteomic screens will be studied in further detail: protein localization survey will assess whether any of these contribute to P granules themselves, and their function will be analyzed by disrupting gene function in vivo, by RNAi, or by expression of dominant-interfering constructs. Conservation of known germ granule components from invertebrates to vertebrates suggests that the results of these studies will advance our understanding of germline development in a broad array of species. In a number of reported cases, disruption of germ cell function results not only in lack of fertility, but also leads to malignant transformations (cancers) in the affected individuals. Advanced knowledge of P granule members and regulators will bring forth improved understanding of both fertility as well as malignancies in humans.